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Fig. 2. 1 H- 15 N HSQC spectra of 15 N-labeled GBAP thiolactone measured at 10°C. Thebackbone amide groups are labeled in capital letters; the side-chain amide and imidegroups in lower-case letters. This peptide has an additional Met residue at the N-terminus.Results and DiscussionThe expression plasmid of the precursor protein of the thiolactone derivative of GBAP inwhich [Cys 3 ]GBAP was fused to the N-terminus of GyrA mini-intein was constructedusing pTXB1 (New England Biolabs). The precursor protein, [Cys 3 ]GBAP-GyrA, was thenexpressed in E. coli Rosetta(DE3) (Novagen) and affinity-purified using chitin resin (NewEngland Biolabs). The [Cys 3 ]GBAP was then cleaved from GyrA mini-intein by treatingthe fusion protein with 2-mercaptoethanesulfonic acid (MESNA) (Figure 1). The resultant[Cys 3 ]GBAP-thioester was autonomously converted to the thiolactone-derivative of GBAP,whose structure and activity were verified by MALDI-TOF-MS and the assay for GBAPactivity, respectively. The activity of the GBAP thiolactone was slightly higher than thenative form of GBAP (lactone). By this method, 0.70 mg and 0.25 mg of non-labeled and15 N-labeled GBAP thiolactones (Figure 2) were purified from 1 L of E. coli culture inTerrific Broth and M9 minimal media, respectively. 1,4-Dithio-DL-threitol (DTT) wastested as an alternative reducing agent to MESNA to cleave [Cys 3 ]GBAP-GyrA, and threeproducts were obtained in this case. Two of them were DTT-adducts to GBAP and theother was the linear form of [Cys 3 ]GBAP. The two different DTT-adducts to GBAP couldbe derived from the optical isomers of DTT. In contrast, no cleavage was observed whentris(2-carboxyethyl)phosphine (TCEP), a non-thiol-type reducing agent, was used.A similar method was proposed to synthesize Staphylococcus aureus autoinducingpeptides (AIPs) from the fusion protein in which each AIP was fused to the N-terminus of amini-intein [5]. This method depends on the autonomously occurring N-S shift-mediatedintein cleavage. However, this method was inefficient in synthesizing GBAP thiolactonedue to low efficiency of autonomous intein cleavage. The intein cleavage in our methodoccurs more efficiently owing to the intermediate thioester formation by MESNA. Thus ourmethod would be more widely applicable to the synthesis of various peptide thiolactones.AcknowledgmentsThis work was supported in part by grants-in-aid for scientific research from the Ministry ofEducation, Culture, Sports, and Technology of Japan (grant 16087203 to K. Nagata), from the JapanSociety for the Promotion of Science (grants 15580065, 17580068 and 40217930 to J. Nakayama),from the Kato Memorial Bioscience Foundation (to J. Nakayama), from the Waksman Foundation ofJapan (to J. Nakayama), and by the Targeted Proteins Research Program (TPRP) of the Ministry ofEducation, Culture, Sports, Science and Technology of Japan (to M. Tanokura).References1. Nakayama, J., et al. Mol. Microbiol. 41, 145-154 (2001).2. Nakayama, J., et al. Biosci. Biotechnol. Biochem. 65, 2322-2325 (2001).3. Nishiguchi, K., et al. J. Bacteriol. 191, 641-650 (2009).4. Evans, T.C. Jr., et al. Protein Sci. 7, 2256-2264 (1998).5. Malone, C.L., et al. Appl. Env. Microbiol. 73, 6036-6044 (2007).163